Genome-wide studies of PAL genes in sorghum and their responses to aphid infestation

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.25) plays a crucial role in plant adaptation to biotic and abiotic stresses. However, the current knowledge about PAL proteins in sorghum is essentially lacking. Thus, in this study we aimed to analyze the PAL family genes in sorghum using a genome-wide approach and to explore the role of PAL genes in host plant resistance to aphids via SA-mediated defense signaling. Here, we report gene structural features of 8 PAL (SbPAL) genes in sorghum (Sorghum bicolor), their phylogeny, protein motifs and promoter analysis. Furthermore, we demonstrated that the SbPAL genes were induced by sugarcane aphid (SCA) infestation and SbPAL exhibited differential gene expression in susceptible and resistant genotypes. PAL activity assays further validated upregulated expression of the SbPAL genes in a resistant genotype. In addition, exogenous application of SA reduced plant damage and suppressed aphid population growth and fecundity in susceptible genotype, suggesting that those SbPAL genes act as positive regulator of the SA-mediated defense signaling pathway to combat aphid pests in sorghum. This study provides insights for further examination of the defense role of PAL in sorghum against other pests and pathogens.

Sugarcane aphid rearing and plant infestation. Sugarcane aphid (SCA) were maintained for generations on Tx7000 plants in greenhouse conditions as described previously 24,36 . Three leaf stage sorghum seedlings were infested with similar-size 20 adults apterous SCA on adaxial surface of the first leaf. Plants were covered individually with cylindrical cages (SABIC Polymershapes, Tulsa, OK) to avoid SCA escape and unwanted pathogen/pest infestation. Plants without SCA infestation were also kept inside a cage as control.
Sequence acquisition and phylogenetic analysis of PAL genes. We obtained the SbPAL amino acid sequence of Sorghum bicolor (sorghum), Brachypodium distachyon (Brachypodium) and Arabidopsis thaliana (Arabidopsis) from phytozome (https:// phyto zome. jgi. doe. gov/ pz/ portal. html). Amino acid sequences of Oryza sativa Japonica Group (rice) and Zea mays (maize) were retrieved from the National Center for Biotechnology Information (NCBI) [http:// www. ncbi. nlm. nih. gov/] and Ensembl (http:// ensem bl. grame ne. org/ Zea_ mays/ Info/ Index) and used as references. The obtained SbPAL sequences were aligned using the MUSCLE program (https:// www. ebi. ac. uk/ Tools/ msa/ muscl e/) 37 . Key amino acids and important conserved sequences were identified by visual observation of SbPAL amino acid sequence alignment. A phylogenetic tree was constructed based on PAL amino acid sequences of rice, maize, wheat, Brachypodium, sorghum, and Arabidopsis, using the maximum likelihood method with poisson correction model (using 1000 boot strap values) in the MEGA7.0 program 38,39 . All DNA and RNA sequences used and analyzed during the current study are available in phytozome (https:// phyto zome. jgi. doe. gov/ pz/ portal. html) and additional data generated from this analysis are available in the supplementary materials/information with this paper.

Identification of conserved motif, cis-element and feature analysis of sorghum PAL genes.
To study structural diversity of SbPALs, conserved motifs were captured using the MEME program (http:// memesuite. org/ tools/ meme) 40 with maximum number of motifs set at 20. To analyze Cis-acting elements, the 2000 base pairs upstream from the ATG start codon of SbPAL genes were first obtained from Phytozome, followed by use of the PLANTRCARE database in the promoter regions 41 . TBtools (toolbox for biologists) v0.6741 was used to generate heat map to visualize expression of cis-acting element related to phytohormones, stress response and lignin biosynthesis in eight SbPAL promoters using the data presented in Supplementary Table S2. ExPASy bioinformatics program (https:// www. expasy. org/) was used to predict Isoelectric point (PI) and molecular weight for each amino acid sequence. Sub-cellular localization of the protein was predicted using CELLO2GO software (http:// cello. life. nctu. edu. tw/ cello 2go). Gene structure display server (GSDS) was used for analysis of exon and intron regions for PAL genes 42 .
RNA extraction and gene expression analysis. Leaf samples were collected from uninfested (control) and infested plants of both genotypes at 6 hours (h), 12 h, 1 days (d), 2d, 3d, 6d and 9d post aphid infestation. The samples were immediately frozen in liquid nitrogen and stored in − 80 °C. Total RNA was extracted from flash-frozen stems and leaves of sorghum plants using TRIzol reagent (Invitrogen), followed by DNase treatment (Turbo DNA free kit, Thermofisher, Waltham, MA) for 30 min at 37 °C. cDNA was synthesized from 1.5 μg of total RNA using the GoScript reverse transcriptase kit (Promega, Madison, WI), following manufacture's instructions. Primers were designed using the IDT DNA program (https:// www. idtdna. com/ Prime rQuest/ Home/ Index), which are listed in Supplementary Table S3. A sorghum β-tubulin gene (Sobic.002g350400) was used as the internal control 43 . Transcript levels were quantified by Quantitative real-time PCR (RT-qPCR) on a PAL activity assay. PAL activity assay was performed using a phenylalanine ammonia-lyase (PAL) microplate assay kit (Cohesion Biosciences) following the manufacturer's instructions. Briefly, fresh 100 mg shoot tissue from uninfested (control) and SCA-infested sorghum plants, respectively, at different timepoints (6 h, 12 h, 1d, 2d, 3d, and 9d) were homogenized with 1 ml of assay buffer in a pre-chilled mortar and pestle. Homogenized samples were centrifuged at 8000× g for 15 min at 4 °C. The reaction mixture was prepared by adding 10 µl supernatant in 130 µl of reaction buffer and 50 µl of L-phenylalanine (substrate) in a 96-well plate, followed by 30 min incubation at 30° C. Stop solution (10 µl) was added to stop the reaction and PAL activity was determined by taking absorption spectra at 290 nm. The observance value from 2 technical and 5 biological replicates were used to calculate PAL activity using the following formula: where one unit (U) is defined as the OD value change of 0.01 per minute; W, the weight of sample in g. The experiment was repeated two times.
Exogenous SA treatment and its effect on SCA infestation in sorghum. Three leaf stage sorghum plants were treated with 50 mL of salicylic acid (SA) [100 ppm] or sterile water per plant by soil-drenching according to Pant et al. 24 , followed by SCA infestation after 24 h as described previously 45 . The second SA treatment was performed after 24 h of SCA infestation, followed by every three days for up to 9 dpi. The number of aphids on SA-treated and non-treated plants were recorded at 2, 3, 6, and 9dpi to determine the role of exogenous SA in aphid defense.

Identification of PAL genes in sorghum and genomic analysis of their structures. To identify
SbPAL gene family members, a homology search using Brachypodium distachyon PAL (BdPAL) amino acid sequences and key word search using "phenylalanine ammonia lyase" was performed against the S. bicolor genome database in phytozome (https:// phyto zome. jgi. doe. gov/ pz/ portal. html). As a result, 8 putative PAL genes were identified from sorghum and their characteristic features were studied (Table 1). Sobic.004G220300 was previously designated as SbPAL1 46 , and names of other genes are here designated as SbPAL2, Sobic.004G220400; SbPAL3, Sobic.004G220500; SbPAL4, Sobic.004G220600; SbPAL5, Sobic.004G220700; SbPAL6, Sobic.001G160500; SbPAL7, Sobic.006G148800; and SbPAL8, Sobic.006G148900. The length of SbPAL proteins varied from 703 (SbPAL7) to 718 (SbPAL2) amino acids, with calculated molecular weights ranging from 75.6 kDa (SbPAL1) to 76.82 (SbPAL3 and SbPAL4). However, sorghum PAL proteins exhibit less variability in isoelectric points (PI) values, indicating that minor variation in ionic strength and/or pH range are required for their optimal activity. Most of the genes (five genes) are clustered on chromosome four, followed by two and one PAL on chromosome six and one, respectively. Subcellular localization of SbPAL proteins was predicted using CELLO2GO software and revealed that all sorghum PAL genes are localized in the cytoplasm (Table 1).

Motifs and gene structural analysis of PAL genes in sorghum.
To gain further understanding about sequence characteristics in SbPAL, we constructed a phylogenetic tree (Fig. 1A)  www.nature.com/scientificreports/ SbPAL4 shared identical amino acid sequence, which is also reflected in the phylogenetic tree. However, they differ in coding sequence as seen in exon-intron structure (Fig. 1B). Furthermore, amino acids sequence analysis also revealed that SbPAL differs in specific amino acid residue at position 123; histidine is present in SbPAL1 and SbPAL7 proteins, tyrosine is present in SbPAL6 whereas phenylalanine is present in rest of the PAL proteins (Supplementary Table 1). In addition, SbPAL6 has an asparagine at residue position 443 whereas lysine is present in the rest of the SbPAL proteins. A previous study stated that PALs with histidine in residue 123 are bifunctional and with phenylalanine in residue position 123 are monofunctional 46 .
To further study structural diversity of SbPAL genes, we predicted exon-intron and motif compositions in coding sequences using the Gene Structural Display Server (http:// gsds. gao-lab. org/). The results showed that five genes possess two exons with varying lengths of intron sequences and rest of the three genes have single exon (Fig. 1B). Interestingly, all genes with single exon were clustered together in the phylogenetic tree ( Fig. 1A). We identified 20 conserved motifs in SbPAL proteins using the MEME suite (http:// meme-suite. org/). The result exhibits that 15 motifs were shared by all PAL proteins, however, rest of the five motifs were uniquely positioned in certain SbPAL (Fig. 1C). For example, motif 20 is uniquely positioned in SbPAL3, SbPAL4, and SbPAL5 and motif 17 is absent in SbPAL1 and SbPAL7. The sequence information of each motif is presented in Supplementary Fig. S1.

Phylogenetic analysis of PAL in sorghum and other plants.
To study the evolutionary relationship of sorghum PAL genes, a total of 51 amino acid sequences from monocotyledon crops (Brachypodium, rice, setaria, maize, sorghum) and the dicotyledon model plant Arabidopsis and apple were used to construct a phylogenetic tree using the maximum likelihood method (with 1000 bootstrap replicates) in MEGA5.0 software (Fig. 2). The phylogenetic tree categorizes all PAL genes into three clusters (Clusters I-III). Furthermore, PAL genes from each of monocot species are clearly distributed across the tree but as expected dicot plants (Arabidopsis and apple) formed a separate group. There are no dicot PAL genes in cluster II, which consists of the members from monocots only (maize, sorghum, and rice) and indicates that these PAL genes might have diverged after separation of monocotyledon and dicotyledon. As mentioned earlier, SbPAL3, SbPAL4 and SbPAL5 are highly similar, and aggregate together within cluster II. Interestingly, SbPAL1 is in cluster I, and is indeed within a same sub-cluster as OsPAL1 and BdPAL1, which are known to be involved in pathogen defense in host plant, suggesting a similar defense function of SbPAL1 in sorghum.

Comparative expression analysis of PAL genes during sugarcane aphid infestation. To get
insight into the role of SbPAL genes during SCA infestation in sorghum, we analyzed their gene expression in shoot tissues from resistant and susceptible sorghum lines using qRT-PCR. We collected shoot tissues from early and late stages of SCA infestation. The samples collected at 6 h, 12 h, 24 h and 48 h were categorized as early stage and those collected at 3d, 6d and 9d after infestation were grouped as late stage. Since SbPAL3 and SbPAL4 are identical in amino acid sequence, we have only selected SbPAL3 for gene expression analysis. The transcript levels of SbPAL6 and SbPAL8 were undetectable or very low in both control and SCA infested samples, hence, were excluded from gene expression analysis. For comparative purposes, the expression of each gene under control conditions was normalized to 1.
In SCA infested samples, SbPAL1, SbPAL2, and SbPAL7 showed a similar trend, and their expressions were significantly induced compared to the uninfested control in the resistant sorghum line at most of the time points of early stage of infestation (Fig. 3A, B). Whereas their expressions in the susceptible line were not significantly increased in the early stage of SCA infestation (Fig. 3). However, their expression on average slowly dropped from  Table 1. www.nature.com/scientificreports/ their peaks at early stage of infestation. At 9 days post infestation (dpi), the expression of SbPAL2 and SbPAL3 genes were significantly elevated again in compared to un-infested plants. We didn't observe any significant induction of SbPAL5 expression in either resistant or susceptible lines at most time points except in the resistant line at 6 dpi and 9 dpi following SCA infestation (Fig. 3D, E). Interestingly, SbPAL1 and SbPAL2 exhibited significant upregulated at 9 dpi in the susceptible line. SbPAL3 showed the highest level of expression at 48 h post infestation (hpi) and consistently showed elevated expression up to 9 dpi in the resistant line. Together, the qRT-PCR analyses revealed that SbPAL genes have differential expression in resistant and susceptible lines, and they are most likely involved in host defense against SCA in a time dependent manner.

Assessment of PAL activity in resistant and susceptible plants.
To determine whether the changes in PAL expression mirrored in PAL activity, we performed PAL activity assays to detect the formation of transcinnamate from L-phenylalanine in SCA susceptible and resistant sorghum lines after 6 h, 12 h, 1d, 2d, 3d and 9d post-infestation with SCA. The results revealed that on average, the SbPAL activities were higher in the resistant line compared to the susceptible line (Fig. 4). Moreover, SCA infestation triggered SbPAL activities in both sorghum lines at all time points.

Analysis of cis-acting elements in SbPAL gene promoter region.
To better understand the transcriptional regulation of SbPALs, we analyzed the promoter region by scanning 2000 bp sequences of the initiation codons of SbPAL genes using the PlantCARE tool 41 (Fig. 5A, B). The PlantCARE tool has predicted dozens of cis-acting elements in SbPAL genes (Supplementary Table S2). Among them, several cis-acting elements belong to phytohormone response and lignin biosynthesis (AC-elements). The cis-acting elements analysis showed that SbPAL genes are mainly regulated by the following phytohormones: methyl jasmonate (MeJA), abscisic acid (ABA), auxin (IAA), gibberellin (GA), and salicylic acid (SA). The sum of the cis-acting elements related to these hormones are presented in Fig. 5A. We predicted several stress-related cis-acting elements that could regulate the SbPAL expression in response to biotic and abiotic stress. The common cis-acting elements related to stress are anaerobic, defense, drought, low temperature, and wound, among which anaerobic-related (ARE) cis-acting elements were the most common, followed by those defense-related elements (STRE and TCrich repeats) and the binding set of the MYB transcription factor (MYB-like sequence) (Fig. 5B). AC-elements which regulated the biosynthesis of lignin, was detected from promoter regions of SbPAL. The AC-elements were found in abundant number in SbPAL1, SbPAL3, SbPAL4, and SbPAL6, indicating their possible role in lignin   Supplementary Table S2.

Effect of exogenous application of SA on host plants during SCA infestations.
PAL is an important enzyme of SA biosynthesis pathway. We have demonstrated via qRT-PCR that PAL expression is induced after SCA infestation in sorghum. It is well established that PAL contributes to activate SA induction in response to biotic stresses 14,20,21,47,48 . We were interested to evaluate if exogenous application of SA could restore susceptibility phenotype of Tx7000 line. For this, we treated plants with SA and studied the plant phenotype, and survival and fecundity of SCA. We found that treatment with SA has a dramatic effect on infestation phenotype, and survival and reproduction in sorghum. Exogenous application of SA partially reduced the SCA susceptible phenotype (e.g., chlorosis, necrosis), which is more pronounced in susceptible lines compared to resistant lines (Fig. 6). In addition, plants treated with SA survived longer compared to untreated plants during the infestation testing when infested with SCA. For instance, the Tx7000 plant usually died two weeks after SCA infestation, whereas after SA treatment, it was able to survive up to three weeks (data not shown). We also counted the number of aphids in the SA-treated and untreated sorghum lines. We found that SA treatment significantly decreased the number of aphids in both resistant and susceptible lines. These differences were more evident in susceptible line, especially in late-stage infestations (9 and 12 dpi) (Fig. 7). Taken together, these results suggest that an induced level of SA plays an important role in resistance to SCA infestation by moderating symptom severity and limiting SCA fecundity.

Discussion
PALs are widely studied plant enzymes in response to biotic and abiotic stresses in plants 1,4,13,20,21 . PALs have also been reported recently, showing their response to aphids in crop plants 11,23,47,49 . In this study, we focused on molecular characterization of the PAL gene family and their functional role in SCA resistance in sorghum using bioinformatics, phenotypic and molecular approaches, and here we reported that SbPAL is involved in host resistance to SCA infestation in sorghum. The PAL enzyme is encoded by a multigene family in plants. The numbers of PAL genes vary in different species, for example, Arabidopsis and tobacco has four PALs 20,50,51 , rice has nine 52 , and Brachypodium has eight 21 . In this study we identified eight genes in sorghum localized in chromosome 1, 4 and 6, with most of them clustered in chromosome 4 ( Table 1). Compared to the number of PAL genes in dicot model plants (Arabidopsis and tobacco), the number of PALs in grass family are higher. The increase in number of genes is considered a natural process of frequent duplication and long evolution. Jun et al. 46 found that amino acid residue at position 123  www.nature.com/scientificreports/ determines specificity to it substrates in SbPAL proteins. Through sequence alignment, we found that at position 123 histidine is present in SbPAL1 and SbPAL7, tyrosine is present in SbPAL6 and phenylalanine is present in the rest of the PAL proteins. PALs with histidine at residue 123 are bifunctional and displayed both PAL and TAL activities, whereas with phenylalanine residue, they are monofunctional and only have PAL activity 46,53 . One bifunctional PAL protein has been identified from Brachypodium (BdPAL1) and maize (ZmPAL1) 8,53 . The variation in number of bifunctional PAL enzymes in closely related grass families suggest an inherent variation among grass species to regulate metabolic flux to respond specific abiotic and biotic stresses 46 . To study whether SbPAL genes are conserved in gene sequences, we performed gene structure and motif analysis. SbPAL2, SbPAL7 and SbPAL8 are intronless and are positioned together in the phylogenetic tree (Fig. 1A). The remaining five SbPALs have single introns and are diverged in the phylogenetic tree based on their varying lengths and sequences of intron. The intron-exon pattern in a gene family reflects the evolutionary history of the gene family 54,55 . In this study, we identified 20 distinct conserved motifs in SbPAL (Fig. 1C), most of which are conserved among the SbPAL family. The results also revealed a few unique motifs whose compositions contribute to the functional diversity of SbPAL genes. The high level of motif conservation in SbPAL suggests conserved biological functions of PAL family members in sorghum. www.nature.com/scientificreports/ The biological function of the SbPAL genes were analyzed in both resistant and susceptible sorghum plants in response to SCA infection at multiple time points. We found different expression patterns among the SbPAL genes, for instances, SbPAL6 and SbPAL8 showed low expression in sorghum shoots and were excluded in this study. Low expression of these two PAL genes were further supported by expression data in the phytozome, which suggested that SbPAL8 is only expressed in roots and SbPAL6 expressed in low levels in the internode region. Expression of remaining genes were significantly induced except SbPAL5 in a time-dependent manner upon exposure to SCA in the resistant line (Fig. 3). However, variance of SbPAL expression level was not significant in the susceptible line. Elevated expression of the SbPAL genes in the resistant sorghum line suggests a strong correlation between upregulation of the SbPAL genes and SCA resistance. Previous reports have shown differential expression of genes belonging to the phenylpropanoid biosynthetic pathway in the susceptible and resistant lines 21,48,56 . Tetreault et al. 56 reported down-regulation of SbPAL genes in the susceptible line (BCK60) at 10-and 15-days post SCA infestation. But Grover et al. 49 showed a suppression of phenylpropanoid proteins on day 1 after SCA infestation, while upregulated on day 7 in a different sorghum line. It is a little inconsistency but is understandable that differential expression of the PAL genes was not consistent among those reports because they were not the same gene though they all belong to the PAL gene family. Apparently, individual genes of the PAL family exhibit very different patterns in response to SCA attack and changes in expression levels at different time points as shown in Fig. 3. This suggests that each member of the PAL gene family plays a different role in the host plant and acts at certain times during the interaction between the host plant and the aphids.
In choice assays, authors showed that SCA has strong preference for the susceptible genotype (BCK60) and have a higher fecundity rate when compared to Tx2783. The authors also found that PALs are localized in cytoplasm and Tx2783 that provide phloem-based resistance, which further confirm other reports about the existence of aphid-resistance factors in phloem 22,57,58 . Pant and Huang 24 also reported higher SCA fecundity in the susceptible line (Tx7000) when compared to the resistance line (Tx2783) which could co-relate to low accumulation of phenylpronoid accumulation along with other resistance factors such as phloem protein coagulation, callose and lectin deposition, and other secondary metabolites [59][60][61][62] . Induction of SbPAL genes and PAL enzyme activity in the resistance line during SCA infestation suggests that the accumulation of phenylpropanoid products in phloem during infestation could have led to the deterrence of SCA.
A strong induction of SbPAL1 at an early stage of infestation suggests its primary defense role in SCA infestation in sorghum. Our in-silico analysis and phylogenetic analysis showed that SbPAL1 and SbPAL7, the bifunctional proteins, are closely associated with Brachypodium bifunctional PAL protein (BdPAL1) and rice PAL (OsPAL1). The role of BdPAL1 in pathogen defense was well documented against Magnaporthe sp., Fusarium www.nature.com/scientificreports/ cuimorum and Panicum mosaic virus 21,48 . Overexpression of OsPAL1 in susceptible variety enhanced resistance to Magnaporthe oryzae 63 . Potential cis-elements associated with stress response like salicylic acid and wounding, and MYB-like sequences, were predicted from promoter regions of SbPAL1, which could drive an overexpression during SCA infestation. For example: An R2R3 MYB transcription factor confers brown plant hopper resistance by inducing expression of the majority of phenylalanine ammonia-lyase genes in rice 64 . Pandey et al. 65 demonstrated that insecticidal proteins were significantly induced under a wound-inducible promoter from rose (RbPCD1pro) in the early stage of Helicoverpa armigera and Myzus persicae infestation, and transgenic plants showed strong resistance against insects. These findings imply SbPAL1 expression was induced early upon SCA infestation in sorghum. However, further studies are needed to understand the functional role of these promoters in SCA resistance in sorghum. The heat map indicated that SbPAL1, SbPAL5, SbPAL7 and SbPAL8 have a higher number of stress responsive cis-elements. Interestingly, SbPAL7 has a greater number of stress responsive and anaerobic stress (ARE)-and wound (WEE3)-responsive and W-box elements, which is consistent with its relatively higher expression in shoots upon SCA infestation (Fig. 3). W-box is a binding site for the WRKY transcription factor and commonly present in the promoter region of PR genes and is induced by SA. A prior study 66 suggested that the W-box plays an important role in SAR and has been identified in promoter regions of PAL genes in cucumber and rice 67 and proposed for their role in SAR and SA-induced defense. Moreover, AC elements are also found from the promoter regions of SbPAL. AC elements are abundant in lignin biosynthesis genes and induce lignin monomer biosynthesis by binding to the MYB transcription factor [68][69][70] . Therefore, higher number of AC elements in promoters of SbPAL1, SbPAL3, SbPAL4 and SbPAL6 suggest that these genes may be involved in the lignin biosynthesis in sorghum.
To further understand the role of SbPAL in SCA resistance in sorghum, a PAL activity assay was performed which affirmed that PAL activity was higher in the resistant line compared to susceptible plants. These findings are aligned with a recent report of proteome analysis in resistant sorghum genotype (SC265), which showed induced expression of PAL protein after 7 days post SCA infestation 49 . These results suggest that PAL enzymes play a crucial role conferring resistance to SCA infestation in sorghum. An elevated level of PAL expression and activity were also commonly observed in other plants during their reactions to pests/pathogens. For example, PAL expression were upregulated in beans, sorghum, Brachypodium, and rice in response to fungal infection 11,21,47 , in Brachypodium and maize to virus infection 23,48 , in Lotus japonicus to Rhizobium symbiosis 71 , in pepper and beans to bacterial pathogens 3,14 and in rice to broad spectrum disease resistance 72 . These studies suggested that the defense role of PALs in plants may be due to accumulation of SA and phenylpropanol intermediates. Furthermore, we investigated whether exogenous application of SA on plants would affect the SCA infestation in susceptible sorghum line. From this study, we demonstrated that the susceptible plant treated with SA alleviated the susceptibility phenotype and significantly reduced the number aphids on the plant compared to SA-untreated plants. Furthermore, SA-treated susceptible plants exhibited delays in the development of the damage symptoms such as chlorosis, necrosis, and subsequent plant death by 5-7 days (Fig. 7). Similar observations were also reported in recent studies, which demonstrated a higher accumulation of SA level in SCA resistant genotype at multiple timepoints 49,73,74 . Furthermore, the recent research results showed upregulation of SA production in sorghum plants following SCA infestation relatively to uninfested plants in both resistant and susceptible genotypes and the levels of SA increased continuously starting from 1-, 3-, through 6-dpi 75 . In the report, the aphid-induced SA production in sorghum plants was also supported by the upregulated expression of SbPAL genes. SA and JA phytohormones behave antagonistically, and defense role of SA in SCA infestation is further supported by a recent study in JA in sorghum 73 . The authors found that JA-deficient sorghum plant reduced aphid feeding and population while exogenous supply of JA attenuated the resistance phenotype and enhanced aphid feeding and population. These observations further support the positive correlation in SA level and aphid resistance in sorghum. Interestingly, exogenous application SA failed to fully restore resistance phenotype to the level of resistant genotype, which entails the existence of multiple resistance factors in phloem in addition to SA.
Taken together, these results suggest that SbPAL confers resistance in sorghum to SCA via SA-dependent defense signaling, which is consistent with the published results 75 . In our results from PAL expression, PAL activity and SA treatment experiments suggest that certain threshold levels in PAL protein and SA were required to confer the resistance to aphid. Susceptible lines failed to meet the minimum threshold level; thus, the plant became susceptible to SCA. However, more studies are required to demonstrate a direct connection between PAL induction and the salicylic acid-mediated defensive response of the plant. Prior studies demonstrated via PAL-silencing experiments in Arabidposis, Brachypodium, maize, and pepper, that PAL plays an important role in SA-dependent signaling of the defense response to plant-pest and pathogen infections 13,14,17,18,21,23 . Thus, these results confirmed that the SbPALs play an important role in SCA resistance in sorghum via SA-dependent defense signaling.

Conclusion
The present study provides the first analysis for motifs, promoters, gene structure, and phylogenetic classification of the PAL gene family in sorghum. Furthermore, our comparative gene expression studies of sorghum in response to SCA between susceptible and resistant lines demonstrated that members of the SbPAL family genes expressed differently during SCA infestation. We showed that SCA infestation in sorghum triggered the induction of SbPAL activation in both susceptible and resistant genotypes. However, strong expression was observed in the resistant line, suggesting that a certain threshold of PAL enzyme activity is required to confer resistance during SCA infestation. A higher PAL enzyme activity in the resistant line further supports that PAL contributes to the host resistance during SCA infestation in sorghum. Exogenous application of SA alleviates the susceptibility phenotype and suppresses the growth and reproduction of SCA in the susceptible line, suggesting an important role of PAL in SCA tolerance via SA-mediated defense signaling. Overall, the findings of our study broaden the